Disulfide conjugated cell toxins and methods of making and using them

ABSTRACT

The invention pertains to the discovery of novel disulfide linked cell toxins which can ablate NK-1 receptor expressing cells. These toxins are used as pharmaceutical compositions for the ablation of NK1 receptor expressing cells and comprise a substance P (SP)- Pseudomonas  exotoxin disulfide linked conjugate. The invention also includes methods of making and using these toxins and pharmaceutical compositions.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims benefit of priority of U.S. ProvisionalApplication No. 60/161,159 filed Oct. 22, 1999, which is hereinincorporated by reference.

FIELD OF THE INVENTION

This invention generally pertains to the field of medicine and paincontrol. In particular, this invention pertains to the discovery ofnovel disulfide linked cell toxins which can ablate neurokinin-1 (NK-1)receptor expressing cells. These toxins are particularly useful aspharmaceutical for the treatment of chronic pain.

BACKGROUND OF THE INVENTION

NK-1 receptor expressing cells are expressed on a variety of cell types.Thus, ablation of these cells can be a means to treat a variety ofcondition of modulate many different physiologic processes. For example,while pain-mediating cells and neurons are the predominant cellsexpressing NK-1 receptors (e.g., spiral cord dorsal horn neurons, seee.g., Basbaum (1999) Reg. Anesth. Pain Med. 24:59-67 ; brain cells, see,e.g., Saria (1999) Eur. J. Pharmacol. 375:51-60; neostriatum cellsthrough the axon collarterals of spiny projection neurons, see, e.g.,Galarraga (1999) Synapse 33:26-35), a variety of other cells alsoexpress NK-1 receptors. Thus, although pain relief is a major result ofthe killing of NK-1 receptor expressing cells, many other conditions canalso be treated. For example, spinal NK-1 receptors modulate autonomicreflexes, including the micturition reflex. In the peripheral nervoussystem, NK-1 receptors are widely expressed in the respiratory,genitourinary and gastrointestinal tracts. NK-1 receptors are alsoexpressed by several types of inflammatory and immune cells. In thecardiovascular system, NK-1 receptors mediate endothelium-dependentvasodilation and plasma protein extravasation. At respiratory level,NK-1 receptors mediate neurogenic inflammation which is especiallyevident upon exposure of the airways to irritants. In the carotid body,NK-1 receptors mediate the ventilatory response to hypoxia. In thegastrointestinal system, NK-1 receptors mediate smooth musclecontraction, regulate water and ion secretion and mediate neuro-neuronalcommunication. In the genitourinary tract, NK-1 receptors are widelydistributed in the renal pelvis, ureter, urinary bladder and urethra andmediate smooth muscle contraction and inflammation in response tonoxious stimuli. NK-1 receptors antagonists, including toxins that canablate NK-1 receptor expressing cells, may have several therapeuticapplications at sites in both the central and peripheral nervous systemsand tissues of the body. In the central nervous system, NK-1 receptorablation toxins could be used to produce analgesia, as antiemetics andfor treatment of certain forms of urinary incontinence due to detrusorhyperreflexia. In the peripheral nervous system, toxins of the inventioncould be used in several inflammatory diseases including arthritis,inflammatory bowel diseases and cystitis (Quartara (1998) Neuropeptides32:1-49). Thus, there exists a need to develop a wide variety of NK-1expressing cell toxins for use as treatments for a variety of differentconditions and as modifiers of a variety of different physiologicmechanisms.

Toxins which kill NK-1 expressing cells can be used to treat pain. Inparticular deman are toxins which can treat chronic pain while at thesame time not significantly affecting the ability to react to acutelypainful, potentially dangerous, stimuli. Efforts to find more effectivetreatments of chronic pain which have few unwanted side effects or whichdo not dampen acutely painful potentially dangerous stimuli remains acontinuing challenge. Current analgesic therapies often fall short oftherapeutic goals and typically have unacceptable side effects. In manychronic pain syndromes, such as those subsequent to neuropathic injury,pain is not well controlled by any currently available method.Furthermore, most chronic pain treatment regimes affect the patient'sability to perceive acute pain, thus blunting or abrogating necessaryprotective basal nociceptive responses. Thus, the discovery of moreefficacious and safe means to control chronic pain is unpredictable andtherapeutically advantageous.

An endogenous peptide ligand of NK-1 receptors is the eleven amino acidlong peptide Substance P (“SP'). SP plays a central role in painsignaling by possibly transducing second messenger signals from primaryafferent nociceptive terminals to second-order neurons in the spinalcord (see, e.g., Lembeck (1981) Neuropeptides 1:175-180). SP transducesa pain signal by interacting primarily with NK-1 receptor-bearing cellsin the brain and spinal chord (see, e.g., Abbadie (1996) Neuroscience70:201-209). Thus, one strategy to control pain has involved making SPantagonists or SP-based toxins that can selectively kill NK-1receptor-bearing cells (see, e.g., Iadarola (1997) Science 278:239-240;Fitzgerald, D., pp. 69 to 82, In Towards a new Pharmacotherapy of Pain,Ed. A. I. Basbaum et al., John Wiley & Sons Ltd. 1991). For example,Goettl, et al. inhibited a pain response in mice by intrathecaladministration of an SP antagonist (Goettl (1998) Brain Res. 780:80-85).Fisher et al. demonstrated that a diptheria toxin-SP recombinant proteinselectively killed cultured cells stably transfected with and expressingNK-1 receptors (Fisher (1996) Neurobiol. 93:7344-7345). One groupinjected rats intrathecally with a saporin toxin-SP disulfide-linkedconjugate. This conjugate killed NK-1 receptor bearing cells in thesuperficial lamina I of the spinal cord and reduced chronic but notacute pain sensation (Mantyh (1997) Science 278:275-279; Wiley (1997)Neurosci. Letters 230:97-100). Benoliel et al. selectively killed NK-1receptor-bearing cells to reduce chronic pain in a rat animal model byintrathecal administration of an SP/diptheria toxin recombinant protein.The recombinant protein killed NK-1 receptor-bearing cells andsignificantly reduced chronic pain as compared to acute pain (Benoliel(1999) Pain 79:243-253). Thus, because of the potential for treating thevery refractive and unpredictable condition of chronic pain, new NK-1receptor bearing cell toxins are needed.

In summary, there exists a need to develop a wide variety of NK-1expressing cell toxins for use as treatments for a variety of differentconditions and as modifiers of a variety of different physiologicmechanisms, particularly in the unpredictable field of chronic paincontrol. It is also extremely important to provide patients with endstage disease proper palliative care and new mediations for control ofsevere intractable pain are needed for this population.

SUMMARY OF THE INVENTION

The invention provides a method of making a cell toxin comprisingreacting a polypeptide comprising a Pseudomonas exotoxin translocationdomain linked to a Pseudomonas exotoxin ADP-ribosylation domain, whereinthe Pseudomonas exotoxin translocation domain comprises at least onereactive sulfhydryl group, with a substance P peptide comprising oneadditional cysteine residue at its amino terminal end, wherein thecysteine sulfydryl group is disulfide linked to a di-thiobis(2-nitro)-benzoic acid group, such that after the reaction a disulfidebond is formed between the exotoxin polypeptide and the substance P anda thionitrobenzoate group is released, and purifying the substanceP-Pseudomonas exotoxin disulfide linked conjugate from the releasedthionitrobenzoate group such that the purified conjugate issubstantially free of thionitrobenzoate groups. In this method thePseudomonas exotoxin translocation domain sulfhydryl group can belocated within ten amino acid residues of the amino terminus or at theamino terminus. The Pseudomonas exotoxin translocation domain sulfhydrylgroup can be a cysteine residue or an equivalent, e.g., apeptidomimetic, an analog, a conservative substitution variation. ThePseudomonas exotoxin translocation domain can be covalently linked tothe Pseudomonas exotoxin ADP-ribosylation domain, for example, thecovalent linkage between the Pseudomonas exotoxin translocation domainand the Pseudomonas exotoxin ADP-riboslyation domain can be a peptidebond, or equivalent structure, as discussed below. The Pseudomonasexotoxin translocation domain can comprise an amino acid sequence as setforth in SEQ ID NO:1. The Pseudomonas exotoxin ADP-riboslyation domaincan comprise an amimo acid sequence as set forth in SEQ ID NO:2.

The invention also provides a pharmaceutical composition for theablation of NK1 receptor expressing cells. The pharmaceuticalcomposition comprises a cell toxin and a pharmaceutically acceptableexcipient, wherein the cell toxin is a substance P-Pseudomonas exotoxindisulfide linked conjugate made by a process comprising the followingsteps: reacting a polypeptide comprising a Pseudomonas exotoxintranslocation domain linked to a Pseudomonas exotoxin ADP-riboslyationdomain, wherein the Pseudomonas exotoxin translocation domain comprisesat least one reactive sulfhydryl group, with a substance P peptidecomprising one additional cysteine residue at its amino terminal end,wherein the cysteine sulfhydryl group is disulfide linked to adi-thiobis (2-nitro)-benzoic acid group, such that after the reaction adisulfide bond is formed between the exotoxin polypeptide and thesubstance P and a thionitrobenozate group is released, and purifying thesubstance P-Pseudomonas exotoxin disulfide linked conjugate from thereleased thionitrobenzoate group such that the purified conjugate issubstantially free of thionitrobenzoate groups. The Pseudomonas exotoxintranslocation domain sulfhydryl group can located within ten amino acidresidues of the amino terminus or it can be located at the aminoterminus. The Pseudomonas exotoxin translocation domain sulfhydryl groupcan be a cysteine residue or equivalent residue, as discussed below. ThePseudomonas exotoxin translocation domain can covalently linked to thePseudomonas exotoxin ADP-riboslyation domain, for example, the covalentlinkage between the Pseudomonas exotoxin translocation domain and thePseudomonas exotoxin ADP-riboslyation domain can be a peptide bond. ThePseudomonas exotoxin translocation domain can comprise an amino acidsequence as set forth in SEQ ID NO:1. The Pseudomonas exotoxinADP-riboslyation domain can comprise an amino acid sequence as set forthin SEQ ID NO:2. The cell toxin and pharmaceutically acceptable excipientcan be suitable for administration intrathecally, subdurally, directlyinto the brain parenchyma, intraventricularly, or directly into a tumor(or systemic administration) for treatment of cancer cells that expressa receptor that binds to SP (e.g., NK-1 receptor).

The invention also provides a method for ablating an NK1 receptorexpressing cell in a patient comprising administering a cell toxin in apharmaceutically acceptable excipient in an amount sufficient to ablatean NK1 receptor expressing cell, wherein the cell toxin is a substanceP-Pseudomonas exotoxin disulfide linked conjugate made by a processcomprising the following steps: reacting a polypeptide comprising aPseudomonas exotoxin translocation domain linked to a Pseudomonasexotoxin ADP-riboslyation domain, wherein the Pseudomonas exotoxintranslocation domain comprises at least one reactive sulfhydryl group,with a substance P peptide comprising one additional cysteine residue atits amino terminal end, wherein the cysteine sulfhydryl group isdisulfide linked to a di-thiobis (2-nitro)-benzoic acid group, such thatafter the reaction a disulfide bond is formed between the exotoxinpolypeptide and the substance P and a thionitrobenzoate group isreleased, and purifying the substance P-Pseudomonas exotoxin disulfidelinked conjugate from the released thionitrobenzoate group such that thepurified conjugate is substantially free of thionitrobenzoate groups.The ablated NK1 receptor expressing cell can be dorsal horn cells,neostriatalcells or other brain parenchyma cells. The ablated NK1receptor expressing cell can also be inflammatory or immune cells,respiratory cells, genitourinary cells or gastrointestinal tract cellsor tumore cells that express a receptor that binds SP (e.g., NK-1receptor).

The invention also provides a method of treating chronic pain withoutsignificantly affecting basal nociceptive responses comprisingadministering a cell toxin in a pharmaceutically acceptable excipient inan amount sufficient to treat chronic pain without significantlyaffecting basal nociceptive responses, wherein the cell toxin is asubstance P-Pseudomonas exotoxin disulfide linked conjugate made by aprocess comprising the following steps: reacting a polypeptidecomprising a Pseudomonas exotoxin translocation domain linked to aPseudomonas exotoxin ADP-ribosylation domain, wherein the Pseudomonasexotoxin translocation domain comprises at least one reactive sulfhydrylgroup, with a substance P peptide comprising one additional cysteineresidue at its amino terminal end, wherein the cysteine sulfhydryl groupis disulfide linked to a di-thiobis (2-nitro)-benzoic acid group, suchthat after the reaction a disulfide bond is formed between the exotoxinpolypeptide and the substance P and a thionitrobenzoate group isreleased, and purifying the substance P-Pseudomonas exotoxin disulfidelinked conjugate from the released thionitrobenzoate group such that thepurified conjugate is substantially free of thionitrobenzoate groups.The cell toxin can be administered, for example, in the form of apharmaceutical composition, to epineurium cells, perineurium cells,nerve ganglia, nerve sheathes, nerve linings, meninges, pia mater cells,arachnoid membrane cells, dura membrane cells, cells lining a joint orbrain or spinal cord parenchymal cells or tumor cells expressing theproper receptor. The pharmaceutical composition can be administeredintrathecally or injected directly into spinal cord or brain parenchymalcells, or joint spaces, or intravenously. For example, thepharmaceutical composition can be delivered into the subarachnoid spacethrough implanted or external intrathecal or epidural pumps.

A further understanding of the nature and advantages of the presentinvention is realized by reference to the remaining portions of thespecification, the figures and claims.

All publications, patents and patent applications cited herein arehereby expressly incorporated by reference for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic summary of data of experiments testing theability of the SP-PE conjugate toxin of the invention to kill CHO cellsstably transfected with and expressing NK-1 receptors (FIG. 1A), NK-2receptors (FIG. 1B), and NK-3 receptors (FIG. 1C), as discussed indetail in Example 1, below.

FIG. 2 shows a schematic summary of data of experiments testing theability of the SP-PE conjugate toxin of the invention to treat pain (asdetermined by a thermal sensitivity test) in an art accepted (rat)animal model for pain control, as explained in detail in Example 3,below.

FIG. 3 shows a schematic summary of data of experiments testing thesegmental selectivity of SP-PE applied at sacral spinal cord.

FIG. 4 shows a schematic summary of data of experiments testing thethermal sensitivity in rats at 46 days following intrathecal injectionof SP-PE conjugate. The ability of the SP-PE conjugate toxin to treatpain effects in an inflammation model of chronic pain was also tested.

DESCRIPTION OF THE INVENTION

This invention is the discovery of a of a novel disulfide linked celltoxin conjugate comprising a modified substance P (“SP”) peptide and amodified Pseudomonas exotoxin polypeptide. This novel cell toxinconjugate is particularly effective in ablating NK-1 receptor-expressingcells. Killing such cells in an effective means of treating a variety ofconditions, particularly, chronic pain or tumors that express a receptorthat binds SP, e.g., NK-1 receptors.

The SP-PE toxins of the invention of particularly efficacious intreating patients with end stage cancer and other long term diseasesassociated with chronic pain who often need greater levels of paincontrol than currently available drugs can provide. Because the SP-PEtoxins of the invention only kill NK-1 receptor expressing cells, theycan be used effectivly in combination, with conventional pain-killingdrugs, e.g., opioids. The Examples below show that the SP-PE toxin ofthe invention only kills substance P receptor (NK-1 receptor)-expressingcells in the spinal cord but spares the opiod receptor expressing cells.Thus, the patient's capacity to be managed with opioids will not beimpaired. Accordingly, the invention provides an advanced and improvedpain management strategy that will provide greater pain relief. Theneeds of these chronic and severe pain patients are underserved and thetoxins of the invention can be used to provide an additional level ofpalliative care by selectively deleting key components in the paintransmission circuit.

Furthermore, in animal model studies, appropriate doses of SP-PE canalso block basal nociceptive responses without overtly impairing otherfunctions such as locomotion. The fact that the toxin of the inventioncan be administered to alleviate pain without detectable motoricinvolvement expands the possible uses of SP-PE, particularly in patientsthat have problems such as cancer pain. For example, the SP-PE toxin canbe used in patients have a chronic disease that causes intermittantproblems with the pain system but chronic pain is always present. Forexample, bone metastasis to the sacral spinal column in prostate cancermakes it very painful to sit and move. This type of “acute” pain can bevery effectively treated with the SP-PE toxins of the invention, whereasother drugs fail routinely.

The modified Pseudomonas exotoxin (“PE”) polypeptide comprises aPseudomonas exotoxin cell translocation domain linked to a Pseudomonasexotoxin ADP-ribosylation domain. The Pseudomonas exotoxin translocationdomain comprises at least one reactive sulfhydryl group; one exemplarytranslocation domain has only one cysteine group (or equivalent, e.g., apeptidomimetic); e.g., at the residue corresponding to position 287 ofthe naturally expressed PE, as described below.

The SP peptide used in the toxin conjugate of the invention comprisesone additional cysteine residue (or equivalent, e.g., a peptidomimetic)at its amino terminal end. This amino terminal residue is disulfidelinked to a di-thiobis (2-nitro)-benzoic acid group, or equivalent.After the reacting the modified PE (with only one active sulfhydrylgroup) and the modified SP together, a disulfide bond is formed betweenthe exotoxin polypeptide and the SP. The reaction also results inrelease of a thionitrobenzoate (or equivalent, if used) group. Thisreaction results in a one to one molar basis between the disulfidelinked SP and PE groups. The SP peptide is also amidated at its carboxyterminal amino acid residue (or equivalent), as discussed below.

The toxin conjugate is then purified from the released thionitrobenzoategroup such that the purified conjugate is substantially free ofthionitrobenzoate groups, i.e., the purified toxin conjugate preparationless than about 2% (on a molar basis) thionitrobenzoate groupsremaining. Thus, the pharmaceutical compositions of the invention haveless than 2% thionitrobenzoate. The invention also comprises toxinconjugate preparations that have less than 1% and 0.5%, and 0.25%“contaminating” thionitrobenzoate (or equivalent, if used) group.

In alternatively embodiments, in place of SP, neuropeptide FF,Neuropeptide Y, preproenkephalin derived peptides, preprodynorphinpeptides, other tachykinin peptides, neurotensin, caclitonin generelated peptide and its fragments can be conjated to PE.

In studies described in the Examples, below, CHO cells stablytransfected with and expressing NK-1 receptors, but not NK-2 or NK-3receptors, were killed by the cell toxin conjugate of the invention.Toxicity to NK-1 receptor expressing cells was blocked by the additionof excess free SP or by treating the toxin conjugate with an anti-SPantibody. Cytotoxicity occurred with exposure times to toxin as short asa two minutes (lethality assessed at 24 hours after exposure). The toxinconjugate of the invention was extremely effective in deleting NK-1receptor expressing cells from the dorsal horn of the spinal cord andNK-1 receptor expressing cells from the striatum. The data described inthe Examples, below, demonstrate that the toxin conjugate of theinvention can kill NK-1 receptor expressing cells, e.g., ablate neuronswhen administered into the cerebral spinal fluid space (CSF) and whenadministered directly into the brain parenchyma.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. As used herein, the following terms havethe meanings ascribed to them unless specified otherwise.

The term “administering” incorporates the common usage and refers to anyappropriate means to give a pharmaceutical to a patient, taking intoconsideration the pharmaceutical composition and the preferred site ofadministration (e.g., in a preferred embodiment, the pharmaceuticalcomposition of the invention is injected into the subarachnoid space asan aqueous solution).

The term “basal nociceptive responses” incorporates its common usage andrefers to baseline responses to nociceptive, or painful, stimuli.

The terms “chronic pain” and “acute pain” incorporate their commonusages; subjective (e.g., clinical diagnosis) and objective means (e.g.,laboratory tests, PET) to determine the presence of chronic pain and/oracute pain, and to distinguish between these two distinct categories ofpain, are described in detail, herein.

The term “pharmaceutically acceptable excipient” incorporates the commonusages and includes any suitable pharmaceutical excipient, including,e.g., water, saline, phosphate buffered saline, Hank's solution,Ringer's solution, dextrose/saline, glucose, lactose, or sucrosesolutions, magnesium stearate, sodium stearate, glycerol monostearate,glycerol, propylene glycol, ethanol, and the like.

The terms “polypeptide” and “peptide” including “Pseudomonas exotoxintranslocation domain, “Pseudomonas exotoxin ADP-ribosylation domain” and“substance P” (or “SP”) peptide includes polypeptides and peptideshaving an activity and a structural characteristic which substantiallycorresponds to their corresponding polypeptides. These include“analogs,” “conservative variants,” “peptidomimetics” and “mimetics”with structures and activity which substantially correspond to exemplarysequences. For example, an exemplary, modified Pseudomonas exotoxintranslocation domain comprises an amino acid sequence as set forth inSEQ ID NO:1 (with only one cysteine residue, as discussed below) and anexemplary Pseudomonas exotoxin ADP-ribosylation domain comprises anamino acid sequence as set forth in SEQ ID NO:2 (residues 400 to 613 ofSEQ ID NO:6). The amino acid sequences set forth in the presentapplication use the conventional practice wherein the amino group ispresented to the left (the N-terminus) and the carboxyl group to theright (the C-terminus) of each amino acid residue.

Human substance P (“SP”) is an eleven amino acid peptide having thesequence RPKPQQFFGLM (SEQ ID NO:3). Naturally occuring SP is amidated onits carboxy terminus. The invention uses a modified twelve residue SPpeptide—a cysteine residue (or equivalent) is added to the aminoterminus; CRPKPQQFFGLM (SEQ ID NO:4), and, the carboxy terminal aminoacid residue (or equivalent) is amidated (in one embodiment, the SP ismade synthetically, amidated, conjugated to TNB and then disulfidelinked to PE). The “polypepties” and “peptides” of the invention include“conservative variants” and “analogs” which have modified amino acidsequences such that the change(s) do not substantially alter thepolypeptide's (the conservative variant's) structure and/or activity.Structural variations to the SP or PE moieties may advantageously alterpotency, selectivity or stability to enzymatic degradation.

Thus, a substance P peptides or variants or mimetic thereof are withinthe scope of the invention if they are capable of binding to an NK-1receptor expressed on a eukaryotic cell, such as a human cell (bindingprotocols for such determinations are well known in the art, see, e.g.,Ciucci (1998) Br. J. Pharmacol. 125:303-401; Maguire (1998) Brain Res.786:263-266). Alternatively, a non-natural residue (e.g.,peptidomimetic) comprising a sulfhydryl or equivalent moiety can bedesigned to replace the cysteine on the N-terminus of the modified SPpeptide used in the toxin conjugates of the invention.

Pseudomonas exotoxin translocation domain variants or mimetics thereofare within the scope of the invention if they are capable oftranslocating through an endosmal/microsmal membrane into the cytosol(means for such determinations are well known in the art, see e.g.,Theuer (1994) Biochemstry 33:5894-5900). A Pseudomonas exotoxinADP-ribosylation domain variant or mimetic thereof is within the scopeof the invention if, after translocation to the cytosol, can inhibitprotein synthesis by ADP-ribosylating elongation factor 2 (means forsuch determinations are well known in the art, see, e.g. Zdanovsky(1993) J. Biol. Chem. 268(29):21791-21799; or, the method of Collier andKandel, as described in Mansfield (1998)Bioconjugate Chem. 7:557-583, p.558). The cell killing effectiveness of the cell toxin can be determinedusing any cytotoxicity assay, many of which are well known in the art(e.g., cell death quantitated using the MTT method of Mosmann (1983) J.Immunol. Meth. 65:55-63).

These include conservatively modified variations of an amino acidsequence, i.e., amino acid substitutions, additions or deletions ofthose residues that are not critical for protein or peptide activity, orsubstitution of amino acids with residues having similar properties(e.g., acidic, basic, positively or negatively charged, polar ornon-polar, etc.) such that the substitutions of even critical aminoacids does not substantially alter structure and/or activity.Conservative substitution tables providing functionally similar aminoacids are well known in the art. For example, one exemplary guideline toselect conservative substitutions includes (original residue followed byexemplary substitution): ala/gly or ser; arg/lys; asn/gln or his;asp/glu; cys/ser; gln/asn; gly/asp; gly/ala or pro; his/asn or gln;ile/leu or val; leu/ile or val; lys/arg or gln or glu; met/leu or tyr orile; phe/met or leu or tyr; ser/thr; thr/ser; trp/tyr; tyr/trp or phe;val/ile or leu. An alternative exemplary guideline uses the followingsix groups, each containing amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Serine (S), Threonine(T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucin(L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); (see also, e.g., Creighton (1984) Proteins, W. H.Freeman and Company; Schulz and Schimer (1979) Principles of ProteinStructure, Springer-Verlag). One of skill in the art will appreciatethat the above-identified substitutions are not the only possibleconservative substitutions. For example, for some purposes, one mayregard all charged amino acids as conservative substitutions for eachother whether they are positive or negative. In addition, individualsubstitutions, deletions or additions which alter, add or delete asingle amino acid or a small percentage of amino acids in an encodedsequence can also be considered “conservatively modified variations.”Routine experimentation will determine whether a conservative variant,analog, mimetic or the like is within the scope of the invention, i.e.,that its structure and/or function is not substantially altered;exemplary means are well known, as described herein.

The terms “mimetic” and “peptidomimetic” refer to a synthetic chemicalcompound which has substantially the same structural and/or functionalcharacteristics of the Pseudomonas exotoxin translocation domain,Pseudomonas exotoxin ADP-ribosylation domain and “SP” polypeptides andpeptides of the invention. The mimetic can be either entirely composedof synthetic, non-natural analogues of amino acids, or, is a chimericmolecule of partly natural peptide amino acids and partly non-naturalanalogs of amino acids. The mimetic can also incorporate any amount ofnatural amino acid conservative substitutions as long as suchsubstitutions also do not substantially alter the mimetic's structureand/or activity. As with polypeptides of the invention which areconservative variants, routine experimentation (as discussed herein)will determine whether a mimetic is within the scope of the invention,i.e., that its structure and/or function is not substantially altered.Polypeptide mimetic compositions can contain any combination ofnonnatural structural components, which are typically from threestructural groups: a) residue linkage groups other than the naturalamide bond (“peptide bond”) linkages; b) non-natural residues in placeof naturally occurring amino acid residues; or c) residues which inducesecondary structural mimiery, i.e., to induce or stabilize a secondarystructure, e.g., a beat turn, gamma turn, beta sheet, alpha helixconformation, and the like.

The term “llinked” means polypeptide domains or individual residues arejoined, “linked” by means other than natural peptide bonds. Theseinclude, e.g., electrostatic (e.g., ionic, van der Waals or hydrogenbonds) or chemical means. For example, the cell toxins of the inventioncomprise a Pseudomonas exotoxin translocation domain linked to aPseudomonas exotoxin ADP-ribosylation domain. Polypeptide domains orindividual peptidomimetic residues can be joined by peptide bonds orother chemical bonds or coupling means, such as, e.g., glutaraldehyde,N-hydroxysuccinimide esters, bifunctional maleimides,N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide(DIC). Linking groups that can be an alternative to the traditionalamide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g.,—C(═O)—CH₂— for —C(═O)—NH—), aminomethylene (CH₂—NH), ethylene, olefin(CH═CH), ether (CH₂—O), thioether (CH₂—S), tetrazole (CN₄—), thiazole,retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp267-357, “Peptide Backbone Modifications,” Marcell Dekker, N.Y.).

A polypeptide can also be characterized as a mimetic by containing allor some non-natural residues in place of naturally occurring amino acidresidues. Nonnatural residues are well described in the scientific andpatent literature; a few exemplary nonnatural compositions useful asmimetics of natural amino acid residues and guidelines are describedbelow. Mimetics of aromatic amino acids can be generated by replacingby, e.g., D- or L-naphylalanine; D- or L-phenylglycine; D- or L-2thieneylalanine; D- or L-1, -2, 3-, or 4-pyreneylalanine; D- or L-3thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- orL-(3-pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- orL-(4-isopropyl)-phenlyglycine; D-(trifluoromethyl)-phenylglycine;D-(trifluoromethyl) -phenylalanine; D-p-fluorophenylalanine; D- orL-p-biphenylphenylalanine; K- or L-p-methoxybiphenylphenylalanine; D- orL-2-indole(alkyl)alanines; and, D- or L-alkylainines, where alkyl can besubstituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentylisopropyl, iso-butyl, sec-isotyl, iso-pentyl, or a non-acidic aminoacids. Aromatic rings of a nonnatural amino acid include, e.g.,thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl,pyrrolyl, and pyridyl aromatic rings. Mimetics of acidic amino acids canbe generated by substitution by, e.g., non-carboxylate amino acids whilemaintaining a negative charge; (phosphono)alanine; sulfated threonine.Carboxyl side groups (e.g., aspartyl or glutamyl) can also beselectively modified by reaction with carbodiimides (R′—N—C—N—R′) suchas, e.g., 1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl)carbodiimide. Aspartyl or glutamylcan also be converted to asparaginyl and glutaminyl residues by reactionwith ammonium ions. Mimetics of basic amino acids can be generation bysubstitution with, e.g., (in addition to lysine and arginine) the aminoacids ornithine, citrulline, or (guanidino)-acetic acid, or(guanidino)alkyl-acetic acid, where alkyl is defined above. Nitrilederivative (e.g., containing the CN-moiety in place of COOH) can besubstituted for asparagine or glutamine. Asparaginyl and glutaminylresidues can be deaminated to the corresponding aspartyl or glutamylresidues. Arginine residue mimetics can be generated by reacting arginylwith, e.g., one or more conventional reagents, including, e.g.,phenylglyoxal, 2,3-butanedoine, 1,2-cyclohexanedione, or ninhydrin,preferably under alkaline conditions. Tyrosine residue mimetics can begenerated by reacting tyrosyl with, e.g., aromatic diazonium compoundsor tetranitromethane. N-acetylimidizol and tetranitromethane can be usedto form O-acetyl tyrosyl species and 3- nitro derivatives respectively.Cysteine residue mimetics can be generated by reacting cysteinylresidues with, e.g., alpha-haloacetates such as 2-chloroacetic acid orchloroacetamide and corresponding amines; to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteine residue mimetics can also begenerated by reacting cysteinyl residues with, e.g.,bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid;chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide;methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3diazole. Lysine mimetics canbe generated (and amino terminal residues can be altered) by reactinglysinyl with, e.g., succinic or other carboxylic acid anhydrides. Lysineand other alpha-amino-containing residue mimetics can also be generatedby reaction with imidoesters, such as methyl picolinimidate, pyridoxalphosphate, pyridoxal, chloroborohydride, trinitro-benzenesulfonic acid,O-methylisourea, 2,4, pentanedione, and transamidase-catalyzed reactionswith glyoxylate. Mimetics of methioninie can be generated by reactionwith, e.g., methionine sulfoxide. Mimetics of proline include, e.g.,pipecolic acid, thiazolidine carboxylic acid, 3- or 4-hydroxy proline,dehydroproline, 3- or 4-methylproline, or 3,3-dimethylproline. Histidineresidue mimetics can be generated by reacting histidyl with, e.g.,diethylprocarbonate or para-bromophenacyl bromide. Other mimeticsinclude, e.g., those generated by hydroxylation of proline and lysine;phosphorylation of the hydroxyl groups of seryl or threonyl residues;methylation of the alpha-amino groups of lysine, arginine and histidine;acetylation of the N-terminal amine; methylation of main chain amideresidues or substitution with N-methyl amino acids; or amidation ofC-terminal carboxyl groups.

A naturally occuring amino acid in a polypeptide or peptide of theinvention can be replaced by a natural or synthetic amino acid ofpeptidomimetic residue of the opposite chirality. Thus, any amino acidnaturally occurring in the L-configuration (which can also be referredto as the R or S, depending upon the structure of the chemical entity)can be replaced with the amino acid of the same chemical structural typeor a peptidomimetic, but the opposite chirality, generally referred toas the D- amino acid, but which can additionally be referred to as theR- or S- form.

The mimetics of the invention can also include compositions that containa structural mimetic residue, particularly a residue that induces ormimics secondary structures, such as a beta turn, beta sheet, alphahelix structures, gamma turns, and the like. For example, substitutionof natural amino acid residues with D-amino acids; N-alpha-methyl aminoacids; C-alpha-methyl amino acids; or dehydroamino acids within apeptide can induce or stabilize beta turns, gamma turns, beta sheets oralpha helix conformations. Beta turn mimetic structures have beendescribed, e.g., by Nagai (1985) Tet. Lett. 26:647-650; Feigl (1986) J.Amer. Chem. Soc. 108:181-182; Kahn (1988) J. Amer. Chem. Soc.110:1638-1639; Kemp (1988) Tet. Lett. 29:5057-5060; Kahn (1988) J.Molec. Recognition 1:75-79. Beta sheet mimetic structures have beendescribed, e.g., by Smith (1992) J. Amer. Chem. Soc. 114:10672-10674.For example, a type VI beta turn induced by a cis amide surrogate,1,5-disubstituted tetrazol, is described by Beusen (1995) Biopolymers36:181-200. Incorporation of achiral omega-amino acid residues togenerate polymethylene units as a substitution for amide bonds isdescribed by Banerjee (1996) Biopolymers 39:769-777. Secondarystructures of polypeptides can be analyzed by, e.g., high-field 1H NMRor 2D NMR spectroscopy, see. e.g., Higgins (1997) J. Pept. Res.50:421-435. See also, Hruby (1997) Biopolymers 43:219-266, Balaji, etal., U.S. Pat. No. 5,612,895.

The skilled artisan will recognize that individual synthetic residuesand polypeptides incorporating mimetics can be synthesized using avariety of procedures and methodologies, which are well described in thescientific and patent literature, e.g., Organic Syntheses CollectiveVolumes, Gilman et al. (Eds) John Wiley & Sons, Inc. N.Y. Polypeptidesincorporating mimetics can also be made using solid phase syntheticprocedures, as described, e.g., by Di Marchi, et al:, U.S. Pat. No.5,422,426. Mimetics of the invention can also be synthesized usingcombinatorial methodologies. Various technique for generation of peptideand peptidomimetic libraries are well known, and include, e.g.,multipin, tea bag, and split-couple-mix techniques; see, e.g., al-Obeidi(1998) Mol. Biotechnol, 9:205-223; Hruby (1997) Curr. Opin. Chem. Biol.1.114-119; Ostergaard (1997) Mol. Divers. 3:17-27; Ostresh (1996)Methods Enzymol. 267:220-234.

The term “pia mater connective tissue” incorporates the common usagerefers to the tissue, membrane, or connective tissue which separates theparenchyma of the spinal cord from the subarachnoid space (the cerebralspinal fluid (CSF) space).

The term “spinal cord parenchymal tissue” incorporates the common usageand refers to the body of the spinal cord containing nerve tissue, e.g.,white and gray mater.

The term “subarachnoid space” or cerebral spinal fluid (CSF) spaceincorporates the common usage refers to the anatomic space between thepia mater and the arachnoid membrane containing CSF.

The term “substantially free of thionitrobenzoate groups” means that thetoxin conjugate is then purified from the released thionitrobenzoate (orits equivalent, if used) group such that the purified conjugate issubstantially free of thionitrobenzoate groups, i.e., the purified toxinconjugate preparation less than about 2% (on a molar basis)thio-nitrobenzoate (or equivalent) groups remaining. Thus, thepharamceutical compositions of the invention have less than 2%thionitrobenzoate. In alternative embodiments, toxin conjugatepreparations (including pharmaceuticals of the invention) that have lessthan 1%, and 0.5%, and 0.25% “contaminating” thionitrobenozoate (orequivalent) group.

The term “treating” refers to any indicia of success in the treatment oramelioration of an injury, pathology, condition, or symptom (e.g.,pain), including any objective or subjective parameter such asabatement; remission; diminishing of symptoms or making the symptom,injury, pathology or condition more tolerable to the patient; decreasingthe frequency or duration of the symptom or condition; slowing in therate of degeneration or decline; making the final point of degenerationless debilitating; improving a patient's physical or mental well-being;or, in some situations, preventing the onset of the symptom orcondition, e.g., pain. The treatment or amelioration of symptoms can bebased on any objective or subjective parameter; including, e.g., theresult of a physical examination and/or a psychiatric evaluation, or,simply an improvement in the patient's sense of well-being. For example,the methods of the invention selectively treats chronic pain byameliorating the hyperalgesia associated with chronic pain, while notsignificantly affecting basal nociceptive responses.

Distinguishing Chronic from Acute Pain

Pain is always subjective and can have physiologic, pathophysiologic,psychologic, emotional, and affective dimensions. Pain causation can bebroadly categorized as organic or psychogenic. Basically, two types ofpain exist—acute pain and chronic pain. Each possibly is mediated byanatomically different nerves. Each type of pain has a differentphysiologic role. For example, the ability to perceive and respond to“acutely” painful stimuli, which usually has the potential to causetissue damage, serves a protective role for the individual. While manytreatments for acute pain cannot ameliorate chronic pain (this, in face,is used as one means to objectively identify “chronic” versus “acute”pain, as discussed below), before this invention, there existed noeffective therapies to treat chronic pain without the unwanted sideeffect of significantly dampening protective acute pain responses.

Diagnosing and Assessing Chronic Pain

The invention provides methods of treating chronic pain while at thesame time not significantly affecting the ability to respond to acutelypainful, and potentially harmful, stimuli. Thus, proper diagnosis ofchronic pain aids in the practice and assessment of efficacy (e.g., fora particular dosage regimen or mode of administration) of thecompositions and methods of the invention. Means to diagnosis chronicpain include classical clinical and psychological evaluations, which canbe augmented by various laboratory procedures, as described herein. Suchmeans as well-described in the medical/scientific and patent literature;some illustrative examples are provided below.

One criteria to diagnose a “chronic” pain is whether the pain persistsfor a month beyond the usual course of an acute disease or a reasonabletime for an injury to heal. This evaluation is made by the clinician ona case by case basis. Acute diseases or injuries can heal in 2, 3, or atmost, 6 weeks, depending on the nature of the condition or injury, theage and health of the patient, and the like. Clinicians are trained tobe very aware of this “acute” versus “chronic” pain distinction, for itis critical to make correct diagnosis and treatment plans. For example,a simple wrist fracture can remain painful for a week to ten days;however, if pain persists longer than this period, a dystropathy couldbe developing which will be irreversible if not treated. See, e.g.,Bonica, et al., (1990) “Management of Pain,” 2nd Ed., Vol. I, Lea &Feibiger, Phil., Pa; Wall and Melzack (1994) “Textbook of Pain,”Churchill Livingston, N.Y. Accordingly, a chronic pain is diagnosed bythe practitioner based on clinical and laboratory results, depending onthe particular condition or injury, patient, and the like (see also,e.g., Russo (1998) Annu. Rev. Med. 49:123-133).

Another means to identify a “chronic” pain is by diagnosis of apathologic process (which is usually also chronic) known to produce orbe associated with chronic pain. Such conditions are well characterizedand include, e.g., chronic pain syndrome (see, e.g., Clifford (1993)Can. Fam. Physician 39:540-559), arthralgia, arthritis (e.g.,osteoarthritis and rheumatoid arthritis), causalgia, hyperpathia,neuralgai, neuritis, radiculagia, fibromyalgia (see, e.g., Simms (1998)Am. J. Med. Sci. 315:346-350), orofacial pain and temporomandibulardisorders (see, e.g., Binderman (1997) Curr. Opin. Periodontol.4:144-15), reflex sympathetic dystrophy (see, e.g., Dangel (1998)Paediatr. Anaesth. 8:105-112, chronic back pain, certain cancers and thelike.

Chronic pain is also associated with particular injuries to the nerves.These include, e.g., nerve transection (traumatic or surgical), chronicabnormal pressure on a nerve, chemical (e.g., formalin) destruction ofnerve tissue, and the like.

Chronic pain can also be distinguished form acute pain by itsnon-responsiveness to pharmacologic therapies known to significantlyameliorate or abate acute pain. When pain is initially diagnosed asacute or of unknown etiology, the clinician typically administers one ofseveral analgesics known in the art to be effective for acute pain, suchas e.g., a non-steroid anti-inflammatory drug (NSAID), such as, e.g.,aspirin, ibuprofen, propoxyphene, tramadol, acetaminophen and the like(see, e.g., Tramer (1998) Acta Anaesthesiol. Scand. 42:71-79). If thereis no significant amelioration of pain, as assessed by the clinician,over an approximately six week period, then a provisional diagnosis ofchronic pain can be made. Ultimately, as discussed above, a diagnosis ofchronic pain depends upon determination as to whether pain would beexpected, given each individual situation.

Other treatments to which chronic pain is also typically incompletely ortotally unresponsive include tricyclic antidepressant administration,psychotherapy, or alternative medicines, such as acupuncture,biofeedback, and the like.

Laboratory, radiographic and other types of imaging procedures can alsobe used to diagnose chronic pain. In particular, positron emissiontomography, or PET, now allows the clinician to objectify such otherwisemerely subjective symptoms, including chronic pain (see, e.g., Reiss(1998) Fortschr. Med. 116:40-43; Di Piero (1991) Pain 46:9-12).

SP Peptides and PE Polypeptides Domains

The invention provides pharmaceutical compositions comprisingpolypeptides cell toxins in an pharmaceutically acceptable excipient.The invention also provides methods of making and using thesepharmaceutical compositions to treat to treat a variety of conditionsresponsive to the ablation of NK-1 receptor expressing cell, e.g.,chronic pain. The invention can be practiced in conjunction with anymethod or protocol known in the art, which are well described in thescientific and patent literature. Therefore, only a few generaltechniques will be described prior to discussing specific methodologiesand examples relative to the novel reagents and methods of theinvention.

GENERAL TECHNIQUES

The polypeptide domains and peptides which comprise the cell toxinconjugates of the invention can be genetically engineered and/orexpressed recombinantly. Techniques for the manipulation of nucleicacids, such as, e.g., subcloning into expression vectors, labelingprobes, sequencing DNA, DNA hybridization are described in thescientific and patent literature, see e.g., Sambrook, ed., MOLECULARCLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring HarborLaboratory, (1989) (“Sambrook”); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,Ausubel, ed. John Wiley & Sons, Inc., New York (1997) (“Ausubel”); and,LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY:HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic AcidPreparation, Tijssen, ed. Elsevier, N.Y. (1993) (“Tijssen”). Productinformation from manufactures of biological reagents and experimentalequipment also provide information regarding known biological methods.Nucleic acids can also be generated or quantitated using amplificationtechniques. Suitable amplification methods include, but are not limitedto: polymerase chain reaction, PCR (PCR PROTOCOLS, A GUIDE TO METHDOSAND APPLICATIONS, ed. Innis, Academic Press, N.Y. (1990), ligase chainreaction (LCR) (Barringer (1990) Gene 89:117); and the like.

Alternatively, peptides and polypeptides domains used to practice theinvention can be chemically synthesized in vitro, whole or in part,using chemical methods well known in the art; see e.g., Caruthers (1980)Nucleic Acids Res. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res.Symp. Ser. 225-232; Banga, A. K., Therapeutic Peptides and Proteins,Formulation, Processing and Delivery Systems (1995) Technomic PublishingCo., Lancaster, Pa. (“Banga”). For example, peptide synthesis can beperformed using various solid-phase techniques (see e.g., Roberge (1995)Science 269:202; Merrifield (1997) Methods Enzymol. 289:3-13) andautomated synthesis may be achieved, e.g., using the ABI 431A PeptideSynthesizer (Perkin Elmer) in accordance with the instructions providedby the manufacturer.

The skilled artisan will recognize that individual synthetic residuesand polypeptides incorporating mimetics can be synthesized using avariety of procedures and methodologies, which are well described in thescientific and patent literature, e.g., Organic Syntheses CollectiveVolumes, Gilman, et al. (Eds) John Wiley & Sons, Inc., N.Y. Polypeptidesincorporating mimetics can also be made using solid phase syntheticprocedures, as described, e.g., by Di Marchi, et al., U.S. Pat. No.5,422,426. Peptides and peptide mimetics of the invention can also besynthesized using combinatorial methodologies. Various techniques forgeneration of peptide and peptidomimetic libraries are well known, andinclude, e.g., multipin, tea bag, and split-couple-mix techniques; see,e.g., al-Obeidi (1998) Mol. Biotechnol. 9:205-223; Hruby (1997) Curr.Opin. Chem. Biol. 1:114-119; Ostergaard (1997) Mol. Divers. 3:17-27;Ostresh (1996) Methods Enzymol. 267:220-234. Modified peptides of theinvention can be further produced by chemical modification methods, see,e.g., Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995)Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry33:7886-7896.

Nucleic acids, peptides and proteins, analogs and mimetics thereof, canbe detected and quantified by any of a number of general means wellknown to those of skill in the art. These include, e.g., analyticalbiochemical methods such as NMR, spectrophotometry, radiography,electrophoresis, capillary electrophoresis, high performance liquidchromatography (HPLC), thin layer chromatography (TLC), andhyperdiffusion chromatogrpahy, various immunological methods, such asfluid or gel precipitin reactions, immunodiffusion (single or double),immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linkedimmunosorbent assays (ELISAs), immunofluorescent assays, Southernanalysis, northern analysis, Dot-blot analysis, gel electrophoresis(e.g., SDS-PAGE), RT-PCR, quantitative PCR, other nucleic acid or targetor signal amplification methods, radiolabeling, scintillation counting,and affinity chromatography.

Pseudomonas exotoxin A

Following SP-NK-1 receptor binding on the cell surface, theligand-receptor complex is internalized. This makes the NK-1 receptor arational target for toxin-derived therapeutics. Pseudomonas exotoxin isa member of a protein family functionally characterized and a “reverseinternalized predator.” Toxins in this class act in the cytosol ofmammalian cells to inhibit protein synthesis and cause cell death atvery low concentrations. These toxins use the endocytosis pathway togain entry to intracellular sorting vesicles, where they unfold andtranslocated into the cytosol. After translocation they refold andenzymatically inhibit protein synthesis. Because of high enzymaticcatalytic rates and apparent resistance of these toxins to proteosomedegradation, only one or a few molecules need to reach to cytosol to beeffective.

Naturally expressed Pseudomonas exotoxin A can be used as a model todesign toxin polypeptide domains to be used in the toxin conjugates ofthe invention. The naturally expressed toxin is secreted by Pseudomonasaeruginosa as a 67 kD protein composed of three prominent globulardomains, termed Ia, II, and III. A small subdomain, termed Ib, connectsdomains II and III (see, e.g., Allured (1986) Proc. Natl. Acad. Sci.83:1320-1324).

Domain Ia of PE mediates cell binding. In nature, domain Ia binds to thelow density lipoprotein receptor-related protein (“LRP”), also known asthe α2-macroglobulin receptor (“α2-MR”) (see, e.g., Kounnas (1992) J.Biol. Chem. 267:12420-23). Domain Ia of PE spans amino acids residues 1to 252. While cell toxin conjugates of the invention lack a functionalPE cell binding domain (replaced by SP in the conjugate), some residuesof this domain may remain in the polypeptide comprising the Pseudomonasexotoxin translocation domain linked to a Pseudomonas exotoxinADP-ribosylation domain.

The Pseudomonas exotoxin translocation domain, or domain II, mediatestranslocation of the polypeptide from the endosome/microsome to thecytosol. Domain II spans amino acids 253 to 364 in naturally occurringPE (see SEQ ID NO:6, below). An amino acid domain sufficient to effecttranslocation can be derived from the translocation domain of native PE.The translocation domain can include the entire sequence of domain II.However, the entire sequence is not necessary for translocation. Forexample, the amino acid sequence can minimally contain, e.g., aminoacids 280-344 (see SEQ ID NO:6, below) of domain II of PE. Sequencesoutside this region, i.e., amino acids 253-279 and/or 345-364, can beeliminated from the domain. This domain also can be engineered withsubstitutions, analogs, mimetic, and the like, so long as translocationactivity is retained. The translocation domain can function as follows.After binding to a receptor on the cell surface, the toxin enters thecell by endocytosis through clathrin-coated pits. In the naturallyexpressed PE, residues 265 and 287 are cysteines that form a disulfideloop. Once internalized into endosomes having an acidic environment, thepeptide is cleaved by the protease furin between Arg279 and Gly280.Then, the disulfide bond is reduced (a mutation at Arg279 inhibitsproteolytic cleavage and subsequent translocation to the cytosol, Ogata(1990) J. Biol. Chem. 265:20678-20685). However, a fragment of PEcontaining the sequence downstream of Arg279 )called “PE37”) retainssubstantial ability to translocate to the cytosol (Siegall (1989) J.Biol Chem. 264:14256-14261). Sequences in domain II beyond amino acid345 also can be deleted without inhibiting translocation. Furthermore,amino acids at positions 339 and 343 appear to be necessary fortranslocation (Siegall (1991) Biochemistry 30:7154-7159). Methods fordetermining the functionality of a translocation domain are well knownin the art, as noted above and described.

Domain Ib of PE has no known function. It spans amino acids 365 to 399of the naturally occurring toxin.

The Psuedomonas exotoxin ADP-ribosylation domain, or domain III, isresponsible for cytotoxicity. The naturally expressed PE includes anendoplasmic reticulum retention sequence which may be retained in thetoxin polypeptide of the invention. Domain III mediates ADP ribosylationof elongation factor 2 (“EF-2”) to inactivate protein synthesis. DomainIII of PE spans amino acids 400 to 613. PE is “non-toxic” if it lacksEF-2 ADP ribosylation activity. Genetically modified forms of PE aredescribed in, e.g., Pastan et al., U.S. Pat. No. 5,602,095; Pastan etal., U.S. Pat. No. 5,512,658 and Pastan et al., U.S. Pat. No. 5,458,878.As noted above, Pseudomonas exotoxin translocation domain andPseudomonas exotoxin ADP-ribosylation domain (and “substance P peptide)include polypeptides having an activity and a structural characteristicwhich substantially corresponds to their corresponding polypeptides,including “analogs,” “conservative variants,” “peptiomimetics” and“mimetics.”

An exemplary Pseudomonas exotoxin translocation domain comprises anamino acid sequence as set forth in SEQ ID NO:1, which has an amino acidsequence the same as residues 280 to 364 of SEQ ID NO:6 (domain Ib hasbeen deleted, see below) except that an initiating methionine is used asresidue in place of the gylcine at residue 280 (in naturally occurringSEQ ID NO:6) (see also Theur (1993) Cancer Res. 53:340-347).

SEQ ID NO:l: Met Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg LeuVal Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val IleArg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile ArgGlu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser GluArg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn

An exemplary Pseudomonas exotoxin ADP-ribosylation domain comprises anamino acid sequence as set forth in SEQ IN NO:2, which corresponds toamino acids 400 to 613 of SEQ ID NO:6.

SEQ ID NO:2: Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly ThrGln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu ArgGly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln Ser IleVal Phe Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg GlyPhe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln GluPro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val ProArg Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro GluAla Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu AspAla Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr Ile Leu Gly TrpPro Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro ArgAsn Val Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala IleSer Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Arg Glu Asp LeuLys

The polypeptides of the invention include conservative variants,mimetics and analogs which have modified amino acid sequences such thatthe change(s) do not substantially alter the variants activity. Thus, aPseudomonas exotoxin translocation domain with conservative variants iswithin the scope of the invention if it is capable of translocatingthrough an endosomal/microsomal membrane into the cytosol (means forsuch determinations are well known in the art, see, e.g., Theuer (1994)supra).

A Pseudomonas exotoxin ADP-ribosylation domain with conservativevariants is within the scope of the invention if, after translocation tothe cytosol, can inhibit protein synthesis by ADP-ribosylatingelongation factor 2 (means for such determinations are well known in theart, see, e.g. Zdanovsky (1993) supra).

The nucleotide sequence (SEQ ID NO:5) and deduced amino acid sequence(SEQ ID NO:6) of Pseudomonas areoginosa exotoxin A are:

GCC GAA GAA GCT TTC GAC CTC TGG AAC GAA TGC GCC AAA GCC TGC GTG 48 AlaGlu Glu Ala Phe Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys Val 1               5                   10                  15 CTC GAC CTCAAG GAC GGC GTG CGT TCC AGC CGC ATG AGC GTC GAC CCG 96 Leu Asp Leu LysAsp Gly Val Arg Ser Ser Arg Met Ser Val Asp Pro             20                  25 GCC ATC GCC GAC ACC AAC GGC CAG GGCGTG CTG CAC TAC TCC ATG GTC 144 Ala Ile Ala Asp Thr Asn Gly Gln Gly ValLeu His Tyr Ser Met Val         35                  40                  45 CTG GAG GGC GGC AACGAC GCG CTC AAG CTG GCC ATC GAC AAC GCC CTC 192 Leu Glu Gly Gly Asn AspAla Leu Lys Leu Ala Ile Asp Asn Ala Leu     50                  55                  60 AGC ATC ACC AGC GAC GGCCTG ACC ATC CGC CTC GAA GGC GGC GTC GAG 240 Ser Ile Thr Ser Asp Gly LeuThr Ile Arg Leu Glu Gly Gly Val Glu65                   70                  75                  80 CCG AACAAG CCG GTG CGC TAC AGC TAC ACG CGC CAG GCG CGC GGC AGT 288 Pro Asn LysPro Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly Ser                 85                  90                  95 TGG TCG CTGAAC TGG CTG GTA CCG ATC GGC CAC GAG AAG CCC TCG AAC 336 Trp Ser Leu AsnTrp Leu Val Pro Ile Gly His Glu Lys Pro Ser Asn            100                 105                 110 ATC AAG GTG TTCATC CAC GAA CTG AAC GCC GGC AAC CAG CTC AGC CAC 384 Ile Lys Val Phe IleHis Glu Leu Asn Ala Gly Asn Gln Leu Ser His        115                 120                 125 ATG TCG CCG ATC TACACC ATC GAG ATG GGC GAC GAG TTG CTG GCG AAG 432 Met Ser Pro Ile Tyr ThrIle Glu Met Gly Asp Glu Leu Leu Ala Lys    130                 135                 140 CTG GCG CGC GAT GCC ACCTTC TTC GTC AGG GCG CAC GAG AGC AAC GAG 480 Leu Ala Arg Asp Ala Thr PhePhe Val Arg Ala His Glu Ser Asn Glu145                 150                 155                 160 ATG CAGCCG ACG CTC GCC ATC AGC CAT GCC GGG GTC AGC GTG GTC ATG 528 Met Gln ProThr Leu Ala Ile Ser His Ala Gly Val Ser Val Val Met                165                 170                 175 GCC CAG ACCCAG CCG CGC CGG GAA AAG CGC TGG AGC GAA TGG GCC AGC 576 Ala Gln Thr GlnPro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala Ser            180                 185                 190 GGC AAG GTG TTGTGC CTG CTC GAC CCG CTG GAC GGG GTC TAC AAC TAC 624 Gly Lys Val Leu CysLeu Leu Asp Pro Leu Asp Gly Val Tyr Asn Tyr        195                 200                 205 CTC GCC CAG CAA CGCTGC AAC CTC GAC GAT ACC TGG GAA GGC AAG ATC 672 Leu Ala Gln Gln Arg CysAsn Leu Asp Asp Thr Trp Glu Gly Lys Ile    210                 215                 220 TAC CGG GTG CTC GCC GGCAAC CCG GCG AAG CAT GAC CTG GAC ATC AAA 720 Tyr Arg Val Leu Ala Gly AsnPro Ala Lys His Asp Leu Asp Ile Lys225                 230                 235                 240 CCC ACGGTC ATC AGT CAT CGC CTG CAC TTT CCC GAG GGC GGC AGC CTG 768 Pro Thr ValIle Ser His Arg Leu His Phe Pro Glu Gly Gly Ser Leu                245                 250                 255 GCC GCG CTGACC GCG CAC CAG GCT TGC CAC CTG CCG CTG GAG ACT TTC 816 Ala Ala Leu ThrAla His Gln Ala Cys His Leu Pro Leu Glu Thr Phe            260                 265                 270 ACC CGT CAT CGCCAG CCG CGC GGC TGG GAA CAA CTG GAG CAG TGC GGC 864 Thr Arg His Arg GlnPro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly        275                 280                 285 TAT CCG GTG CAG CGGCTG GTC GCC CTC TAC CTG GCG GCG CGG CTG TCG 912 Tyr Pro Val Gln Arg LeuVal Ala Leu Tyr Leu Ala Ala Arg Leu Ser    290                 295                 300 TGG AAC CAG GTC GAC CAGGTG ATC CGC AAC GCC CTG GCC AGC CCC GGC 960 Trp Asn Gln Val Asp Gln ValIle Arg Asn Ala Leu Ala Ser Pro Gly305                 310                 315                 320 AGC GGCGGC GAC CTG GGC GAA GCG ATC CGC GAG CAG CCG GAG CAG GCC 1008 Ser Gly GlyAsp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala                325                 330                 335 CGT CTG GCCCTG ACC CTG GCC GCC GCC GAG AGC GAG CGC TTC GTC CGG 1056 Arg Leu Ala LeuThr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg            340                 345                 350 CAG GGC ACC GGCAAC GAC GAG GCC GGC GCG GCC AAC GCC GAC GTG GTG 1104 Gln Gly Thr Gly AsnAsp Glu Ala Gly Ala Ala Asn Ala Asp Val Val        355                 360                 365 AGC CTG ACC TGC CCGGTC GCC GCC GGT GAA TGC GCG GGC CCG GCG GAC 1152 Ser Leu Thr Cys Pro ValAla Ala Gly Glu Cys Ala Gly Pro Ala Asp    370                 375                 380 AGC GGC GAC GCC CTG CTGGAG CGC AAC TAT CCC ACT GGC GCG GAG TTC 1200 Ser Gly Asp Ala Leu Leu GluArg Asn Tyr Pro Thr Gly Ala Glu Phe385                 390                 395                 400 CTC GGCGAC GGC GGC GAC GTC AGC TTC AGC ACC CGC GGC ACG CAG AAC 1248 Leu Gly AspGly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn                405                 410                 415 TGG ACG GTGGAG CGG CTG CTC CAG GCG CAC CGC CAA CTG GAG GAG CGC 1296 Trp Thr Val GluArg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg            420                 425                 430 GGC TAT GTG TTCGTC GGC TAC CAC GGC ACC TTC CTC GAA GCG GCG CAA 1344 Gly Tyr Val Phe ValGly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln        435                 440                 445 AGC ATC GTC TTC GGCGGG GTG CGC GCG CGC AGC CAG GAC CTC GAC GCG 1392 Ser Ile Val Phe Gly GlyVal Arg Ala Arg Ser Gln Asp Leu Asp Ala    450                 455                 460 ATC TGG CGC GGT TTC TATATC GCC GGC GAT CCG GCG CTG GCC TAC GGC 1440 Ile Trp Arg Gly Phe Tyr IleAla Gly Asp Pro Ala Leu Ala Tyr Gly465                 470                 475                 480 TAC GCCCAG GAC CAG GAA CCC GAC GCA CGC GGC CGG ATC CGC AAC GGT 1488 Tyr Ala GlnAsp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly                485                 490                 495 GCC CTG CTGCGG GTC TAT GTG CCG CGC TCG AGC CTG CCG GGC TTC TAC 1536 Ala Leu Leu ArgVal Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe Tyr            500                 505                 510 CGC ACC AGC CTGACC CTG GCC GCG CCG GAG GCG GCG GGC GAG GTC GAA 1584 Arg Thr Ser Leu ThrLeu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu        515                 520                 525 CGG CTG ATC GGC CATCCG CTG CCG CTG CGC CTG GAC GCC ATC ACC GGC 1632 Arg Leu Ile Gly His ProLeu Pro Leu Arg Leu Asp Ala Ile Thr Gly    530                 535                 540 CCC GAG GAG GAA GGC GGGCGC CTG GAG ACC ATT CTC GGC TGG CCG CTG 1680 Pro Glu Glu Glu Gly Gly ArgLeu Glu Thr Ile Leu Gly Trp Pro Leu545                 550                 555                 560 GCC GAGCGC ACC GTG GTG ATT CCC TCG GCG ATC CCC ACC GAC CCG CGC 1728 Ala Glu ArgThr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg                565                 570                 575 AAC GTC GGCGGC GAC CTC GAC CCG TCC AGC ATC CCC GAC AAG GAA CAG 1776 Asn Val Gly GlyAsp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln            580                 585                 590 GCG ATC AGC GCCCTG CCG GAC TAC GCC AGC CAG CCC GGC AAA CCG CCG 1824 Ala Ile Ser Ala LeuPro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro        595                 600                 605 CGC GAG GAC CTG AAG1839 Arg Glu Asp Leu Lys     610

Assessing Translocation to the Cytosol

Any functional Pseudomonas polypeptide translocation domain can be usedin the toxin conjugate of the invention. The functionality of atranslocation domain can be tested as a function of the domain's abilityto mediate and gain access to the cytosol (because access first requiresbinding to the cell, these assays are also useful to determine whetherthe SP domain is functioning, i.e., binding to an NK-1 expressing cell).In one method, access to the cytosol is determined by detecting thephysical presence of the toxin conjugate in the cytosol. For example,the toxin conjugate can be labeled with a composition that is detectableafter the toxin conjugate gains entry inside a cell. Then, the cytosolicfraction is isolated (the endosomal compartment can also be separatelyisolated) and the amount of label in the cytosol fraction determined.Detecting label in the cytosol indicates that the translocation domainis functional and that the toxin conjugate has gained access to thecytosol.

Assessing ADP Ribosylation Activity

Any functional Pseudomonas polypeptide translocation domain can be usedin the toxin conjuate of the invention. The functionality of a domainIII having ADP ribosylation activity can also be tested in a variety ofways. For example, cells can be seeded in tissue culture plates andexpoed to the toxin conjuate. ADP ribosylation activity is determined asa function of inhibition of protein synthesis by, e.g., monitoring theincorporation of ³H-leucine.

Recombinant Expression of Pseudomonas Polypeptide Domains

The functional Pseudomonas polypeptide translocation and ADPribosylation domains can be expressed in any in vitro or in vivorecombinant system. In one exemplary system, Pseudomonas polypeptidetranslocation and ADP ribosylation domains are designed as a singlerecombinant molecule based on the naturally expressed toxin (SEQ IDNO:6). In one exemplary design the recombinant molecules uses aninitiating methionine in place of glycine at residue 280 (of SEQ IDNO:6), retains the cysteine of residue position 287, and eliminates thedisulfide bond at residues 374 to 379; the resulting polypeptide is SEQID NO:1.

In one exemplary recombinant system, SEQ ID NO:1 (or equivalent thereof)is expressed into the periplasm of E. coli (using standard techniques,see, e.g., Sambrook). There, a large proportion of the toxin dimerizesby forming a disulfide bond (linking cysteins 287). Prior toconjugation, PE recombinant polypeptide preparations are treated with2-mercaptoethanol and separated on a size exclusion chromatographycolumn (using standard techniques). Any remaining dimers elute first,followed by monomer fractions, which are appropriately collected forfurther conjugation to SP-thionitrobenzoic acid derivatized peptide, asdescribed below.

Substance P and the NK-1 Receptor

An important pain-mediating peptide is the eleven amino acid longSubstance P (“SP”). SP plays a central role in the transduction ofsecond messenger signals from primary afferent nociceptive terminals tosecond-order neurons in the spinal cord (see, e.g., Lembeck (1981)supra). SP transduces a pain signal by interacting primarily withneurokini-1 (“NK1”) receptor-bearing cells in the brain and spinal chord(see, e.g., Abbadie (1996) supra; Seelig (1996) Biochemistry35:4365-4374). One of the most studied SP circuits is the connectionbetween primary afferent nociceptive dorsal root ganglion neurons andsecond order neurons in the spinal cord. This SP mediated signal isknown to mediate pain signals. These second order neurons are essentiallinks in the classical trisynaptic pathway of pain transmission from theperiphery to the spinal cord to the thalamus to the somatosensory are ofthe cortex.

An SP peptide or variant thereof is within the scope of the invention ifit is capable of binding to an NK-1 receptor expressed on a eukaryoticcell, such as human cell (binding protocols for such determinations arewell known in the art, see, e.g., Ciucci (1998) Br. J. Pharmacol.125:393-401; Maguire (1998) Brain Res. 786:263-266; Quartara (1998)supra) and discussion below. SP peptide used in the toxin conjugates ofthe invention are further modified by the addition of a sulfhydrylgroup, e.g., a cysteine residue or equivalent, to the amino terminus ofthe peptide (for disulfide linked conjugation, as discussed below).Additional of a sulfhydryl group to the amino terminus is importantbecause the carboxy-terminus of the SP peptide must be amidated for thepeptide to bind to the NK-1 receptor. SP peptides used in the inventioncan be amidated on their carboxy termini by any means, e.g., byamidation of peptides which have been made synthetically or by themethod set forth in Fisher (1996) supra (page 7342).

The invention provides a pharmaceutical composition and a method for theablation of NK1 receptor expressing cells. Thus, the cell toxins of theinvention can bind to (and kill) any NK1 receptor expressing cell. Whilepain mediating cells and neurons are the predominant cells expressingNK-1 receptors (e.g., spinal cord dorsal horn neurons, see, e.g.,Basbaum (1999) supra), brain cells, see, e.g., Saria (1999) supra;neostriatum cells through the axon collaterals of spiny projectionneurons, see, e.g., Galarrga (1999) supra), a variety of other normal orabnormal (e.g., tumor) cells also express NK-1 receptor and thus can bekilled practicing this invention. Thus, the cell toxins and methods ofthe invention can be used to treat a variety of different conditions orto modify a variety of different physiologic mechanisms. For example,spinal NK1 receptors modulate autonomic reflexes, including themicturition reflex. In the peripheral nervious system, NK1 receptors arewidely expressed in the respiratory, genitourinary and gastrointestinaltracts and are also expressed by several types of inflammatory andimmune cells. In the cardiovascular system, NK1 receptors mediateendothelium-dependent vasodilation and plasma protien extravasation. Atrespiratory level, NK1 receptors mediate neurogenic inflammation whichis especially evident upon exposure of the airways to irritants. In thecarotid body, NK1 receptors mediate the ventilatory response to hypoxia.In the gastrointestinal system, NK1 receptors mediate smooth musclecontraction, regulate water and ion secretion and mediate neuro-neuronalcommunication. In the genitourinary tract, NK1 receptors are widelydistributed in the renal pelvis, ureter, urinary bladder and urethra andmediate smooth muscle contraction and inflammation in response tonoxious stimuli. NK1 receptors antagonists, including toxins that canablate NK-1 receptor expression cells, may have several therapeuticapplications at central and peripheral level. In the central nervoussystem, NK1 receptor ablation toxins could be used to produce analgesia,as antiemetics and for treatment of certain forms of urinaryincontinence due to detrusor hyperreflexia. In the peripheral nervoussystem, toxins of the invention could be used in severl inflammatorydiseases including arthritis, inflammatory bowel diseases and cystitis(Quartara (1998) supra). Further uses of the toxins and methods of theinvention are suggested by, e.g., Al, et al., who show that SP and itsreceptor are expressed in human peripheral blood-isolated lymphocytes(Lai (1998) J. Neuroimmunol. 86:80-86). Using cultured rabbitosteoclasts, Mori et al. found that SP possibly stimulates the boneremodeling by osteoclasts (Mori (1999) Biochem. Biophys. Res. Commun.262:418-422). SP present in bronchial nerve fibers can induce relaxationof rat bronchial smooth muscle (Bodelsson (1999) Respiration66:355-359). SP has aniolytic-like effects when administered into thenucleus basalis of the rat ventral pallidum (Nikolaus (1999) Neuroreport(10:2293-2296). Baraniuk, et al. showed that a nociceptive nerveefferent axon response may lead to glandular exocytosis through actionson submucosal gland NK-1 receptors (Baraniuk (1999) Am. J. Respir. Crit.Care Med. 160:655-662). Mauback, et al. showed that a SP receptorantagonist had antidepressant efficacy (Maubach (1999) Curr. Opin. Chem.Biol. 3:481-488). Santoni, et al. speculates that SP plays a major rolein the regulation of the interaction between immuned and nervoussystems. Santoni (1999) J. Neuroimmunol. 93:15-25).

The conjugates may also be used in the treatment of neurologicaldysfunctions of the basal ganglia by targeting cholinergic interneuronsthat express SP (e.g. Parkinsons Disease, see, e.g., Kaneko Science289:633-637, 2000). In such a use, the compostion is typicallyadministered directly to the brain.

Disulfide Linked Cell Toxin Conjugates

The invention provides disulfide linked cell toxin conjugates comprisinga modified SP peptide and a modified PE translocation domain linked to aPE ADP-ribosylation domain. chemical conjuation is advantageous oversynthesis by, e.g., entirely recombinant techniques, because a betterdefined homogenous product is obtained (recombinant expression in vivo,e.g., mammalian cells or bacteria, in contrast, commonly results inmixed products due to posttranslational modification, degradation, andthe like). In the present invention, conjugates are made by “disulfideexchange.” To ensure that the ratio of SP to PE in each conjugatemolecule is 1:1, the SP peptide and the Pseudomonas exotoxin componentof the toxin conjugate each have only one reactive sulfhydryl group. Thesulfhydryl group for disulfide exchange can be in the form of a cysteine(or equivalent, see above) residue.

To make the conjugate, the SP-cysteeine (or equivalent) (e.g., SEQ IDNO:4), generated by solid phase synthesis, initially purified, andamidated. It next reacted with dithio-bis(2-nitro)-benzoic acid (DTNB).This reaction yields a thionitrobenzoic acid derivatized SP peptide(“SP-TNB”) and a thionitro benzoate group.

In one exemplary protocol, SP-TNB is added to the pseudomonas exotoxincomponent (having only one reactive sulfhydryl group) in a 8:1 molarratio for an overnight incubation at 4C. Progress of the reaction ismonitored, e.g., by measuring absorbance at OD 412 nm. SP-TNB can bepurified, e.g., by HPLC. The SP-PE pharmaceuticals of the invention haveless than 2% thionitro benzoate groups.

SP-TNB is dissolved in 20% DMSO (e.g., 2 mg SP-TNB in 20 microlitersDMSO). Then 1.0 milliliter of 0.2 molar Na-phosphate is added (pH 7.0).The conjugation mixture is applied to gel filtration column (e.g., G-25)to remove unreacted SP-TNB and eluted. The mixture was fixture purifiedby HPLC gel filtration to remove unreacted PE polypeptide. Fractions arecollected and analyzed by Western blot to confirm the presence ofPseudomonas exotoxin domain (by reaction with monoclonal anti-PEantibody) and monoclonal anti-SP antibody (directed to the amidatedcarboxy terminus of the SP). The presence of a disulfide linkage isconfirmed by reduction (e.g., with 2-ME or dithiothreito) followed bygel electrophoresis (e.g., SDS-PAGE) and Western blot analysis with theabove referenced monoclonal antibodies. Fractions containing one to one(1:1) PE to SP molar relationship conjugates are retained and stored,e.g., at −80° C.

Formulation and Administration Pharmaceuticals

The cell toxin conjugates of the invention are formulated aspharmaceuticals to be used in the methods of the invention to treatpain, particularly chronic pain. These pharmaceuticals can beadministered by any means in any appropriate formulation. Routine meansto determine drug regimens and formulatins to practice the methods ofthe invention are well described in the patent and scientificliterature, and some illustrative examples are set forth below. Forexample, details on techniques for formulation and administration arewell described in the scientific and patent literature, see, e.g., thelatest edition of Remington's Pharmaceutical Sciences, Maack PublishingCo, Easton, Pa.

The pharmaceutical composition of the invention is administered suchthat the cell toxin conjugate is anatomically approximate to an NK-1recetor bearing cell, e.g., a nerve, including, e.g., epineurium orperineurium, tissue surrounding nerve ganglia, nerve sheathes, nervelinings, or meninges, e.g., the pia mater, or arachnoid or duramembranes. The pharmaceutical composition can be administered into thesubarachnoid space. The pharmaceutical composition can be administeredin or approximate to joints for, e.g., the treatment of chronic painassociated with arthritis.

The pharmaceutical composition can be administered intrathecally (i.e.,into the CSF in the subarachnoid space), where the concentration of celltoxin conjugate in the pharmaceutically acceptable excipient can bebetwwen about is 0.5 to about 50 mL of a formulation at dosagesequivalent to about 0.2 to about 0.6 mg/kg, or, about 0.1 to about 1.0mg/kg, or, about 0.2 to 10³ nanograms per microliter, depending on avariety of conditions, as described below. The delivery can be throughimplanted or external intrathecal or epidural pumps, see, e.g.,Hassenbusch (1999) Oncology 13)5 Suppl 2):63-7.

Aqueous suspensions of the invention can also include any excipients oradmixture suitable for the manufacture of aqueous suspensions. Theaqueous suspension can also contain one or more preservatives, such asethyl or n-propyl p-hydroxybenzoate. The aqueous suspeonsion can beadjusted for osmolarity. The aqueous solution can be adjusted to promotethe stability of the vector for lyophilization. For example, acryoprotectant solution that can significantly maintain stability afterfreeze-thaw cycles.

The pharmaceutical formulations of the invention can be provided as asalt and can be formed with many acids, including but not limited tohydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc.Salts tend to be more soluble in aqueous or other protonic solvents thatare the corresponding free base forms. In other cases, the preparationmay be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose,2%-7% mannitol at a pH range of 4.5 to 5.5, that is combined with theappropriate aqueous buffer prior to use.

After a pharmaceutical comprising a cell toxin conjugate of theinvention has been formulated in an acceptable carrier/excipient it canbe placed in an appropriate container, e.g., as a kit, and labeled fortreatment of the indicated condition. for administration of thepharmaceutical compositions of the invention, such labeling wouldinclude, e.g., instructions concerning the amount, frequency and methodof administration. These instructions can also be part of a kit.

The cell toxin conjugates of the invention can also be formulated ascationic lipid-nucleic acid compositions for administration in a varietyof ways. Details on techniques for formulation and administration arewell described in the scientific and patent literature, see, e.g., thelatest edition of “Remington's Pharmaceutical Sciences” (MaackPublishing Co, Easton Pa.). See also, Lasic and Templeton (1996) Adv.Drug Deliv. Rev. 20:221-266 and references cited therein. The ratios ofeach component in the cationic lipid-nucleic acid complexes, finalconcentrations, buffer solutions, and the like can be readily optimizedby the skilled artisan, taking into consideration the mode of delivery(i.e., intrathecal, epidural, intra-articular, direct injection intobrain or spinal cord parenchyma), the anatomical site of delivery, anyexistent conditons or diseases, the condition and age of the patient,and the like. Methods of producing cationic liposomes are known in theart (see e.g., Liposome Technology (CFC Press, N.Y. 1984); Liposomes,Ortro (Marcel Schler, 1987).

Determining Dosing Regiments

The pharmaceutical formulations of the invention can be administered ina variety of unit dosage forms, depending upon the particular conditionor disease, the degree of chronic pain, the general medical condition ofeach patient, the method of administration, and the like. Details ondosages are well described in the scientific and patent literature, see,e.g., the lastest edition of Remington's Pharmaceutical Sciences, MaackPublishing Co, Easton Pa.

The exact amount and concentration of cell toxin conjugate and theamount of formulation in a given dose, or the “therapeutically effectivedose” is determined by the clinician, as discussed above. The dosageschedule, i.e., the “dosing regimen,” will depend upon a variety offactors, including, e.g., the amount of chronic pain present, theduration of the pain, the stage and severity of the disease or conditionassociated with the chronic pain (if any), and the general state of thepatient's health, physical status, age and the like. The state of theart allows the clinician to determine the dosage regimen for eachindividual patient and, if appropriate, concurrent disease or conditiontravel, see, e.g. Selvaggi (1993) J. Immunother, 13:201-207.

One typical dosage is between about 0.5 to about 50 mL of a formulationat dosages equivalent to about 0.2 to about 0.6 mg/kg daily (however,the dosage can be adjusted, as described below, from about 0.01 to about2.0 mg/kg). Dosage levels and frequency of administration (daily forseveral days, every other day, etc.) are based on objective andsubjective criteria, as discussed herein. Any dosage can be used asrequired and tolerated by the patient. Furthermore, the exactconcentration of toxin conjugate, the amount of formulation, and thefrequency of administration can be adjusted depending on the levels ofSP-PE measured in the CSF after an initial administration. Means tosample CSF or other fluid or tissue samples and detect and quantitatelevels of SP-PE are well known in the art (see Example 1, below).

Routes of Administration

The pharmaceutical compositions of the invention can be administered byany means know in the art to any anatomical space, e.g., intrathecally,subdurally, direct injection in brain or spinal cord parenchyma. Iftreating a cancer that expresses a receptor that binds to SP (e.g. NK-1receptor), the pharmaceutical can also be administered, e.g.,intravenously or directly into the tumor. The pharmaceutical compositionof the invention can be administered to any cell which expresses NK-1receptors, including, but not limited to epineurium cells, perineuriumcells, nerve ganglia, nerve sheathes, nerve linings, meninges, pia matercells, arachnoid membrane cells, dura membrane cells, cells lining ajoint or brain or spinal cord parenchymal cells. For example, thepharmaceutical composition can be administered intrathecally orepidurally in a pharmaceutically acceptable excipient. Means toadminister solutions into all of these anatomical compartments are wellknown in the art, see, e.g., the subarachnoid space, i.e.,intrathecally, into the CSF, see, e.g., Oyama, T., U.S. Pat. No.4,313,937; discussing intratecal pumps, see, e.g., Nance (1999) Phys.Med. Rehabil. Clin. N. Am. 10:385-401; Anderson (1999) Neurosurgery44:289-300.

The cell toxin of the invention can be administered by single ormultiple or continuous infusion (e.g., by pumps, internal or external)into the intrathecal or epidural space, or directly into brain or spinalcord parenchyma, depending on the dosage and frequency as required andtolerated by the patient.

Kits

The invention provides a kit for the treatment of chronic pain in ahuman which includes a pharmaceutical composition of the invention. Thekit can contain instructional material teaching preferred indications,dosages and schedules of administration, and the like.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

EXAMPLES

The following example is offered to illustrate, but not to limit theclaimed invention.

Example 1 Substance P-Pseudomonas Exotoxin Disulfide Linked ConjugateSelectively and Potently Kills Neurokinin-1 Receptor Expressing Cells

The followng example details a study which demonsrates that compositionsand methods of the invetnion can be practiced to ablate NK-1 receptorexpressing cells. Specifically, this study demonstrates that CHO cellsstably transfected with and expressing NK-1 receptors, but not NK-2 orNK-3 receptors, can be killed by the SP-PE cell toxin conjugate of theinvention.

A disulfide linked SP-PE cell toxin was made as described above.Specifically, an synthetic SP peptide (SEQ ID NO:4) was carboxy-terminalamidated prior to conjuation using standard techniques.

The carboxy-terminal amidated, N-terminal thio-nitrobenzoic acidderivatized SP peptide was reacted with a recombinant PE polypeptidehaving a N-terminal domain with a sequence as set forth in SEQ ID NO:1and a carboxy-terminal domain (the translocation domain) with a sequenceas set forth in SEQ ID NO:2 ) the ADP-ribosylation domain). The celltoxin conjugate was purified and analyzed for the presence of disulfidelinkage, SP peptide and PE domains as describe above. The preparationwas substantially pure of free thionitrobenzoate groups.

CHO cells were stably transfected with either NK-1, NK-2 or NK-3receptor encoding nucleic acid sequences (see, e.g., Maggi (1997) TrendsPharmacol. Sci. 18:351-355; Tian (1996) J. Neurochem. 67:1191-1199)using conventional techniques. Cell surface expression of eachrespective receptor was confirmed by reaction with receptor specificantibodies (see, e.g., Dery (1997) J. Neuroimmunol. 76:1-9); Arkinstall(1995) FEBS Letters 375:183-187.

NK-1, NK-2, or NK-3 receptor-expressing CHO transformant cell cultureswere grown to 90% confluence. The cultures were exposed to the SP-PEcell toxin for various times, including 1.5 minutes, 3 hours, and 24hours. After exposure, cells were washed and allowed to proliferate fora further 24 or 48 hours. At these times media and an non-adherent cells(i.e., dead cells) were removed and the cultures fixed and stained withtoluidine/methylene blue in ethanol. These stained cultures werephotographed and the images on the slides were digitized by scanningwith a digital camera. The average number of live/dead cells per platewas calculated by using the “analyze” and “histogram” functions inADOBE™ photoshop program using standard techniques.

The SP-PE toxin was very specific for NK-1 receptor-expressing cells,which were killed with an IC50 of 2 ng/ml (5×10⁻¹¹ M). No killing wasseen when NK-1 receptor-expressing cells were incubated withunconjugated PE domains. No killing was seen when NK-2 or NK-3receptor-expressing cells were exposed to SP-PE toxin.

Toxicity to NK-1 receptor expressing cells were blocked by the additionof excess free SP or by treating the toxin conjugate with an anti-SPantibody. Cytotoxicity occurred with exposure times to toxin as short atwo minutes (lethality assessed at 24 hours after exposure).

The specificity for SP-PE toxin for NK-1 receptor-expressing cells wasconfirmed by incubating the panel of stably transfected cells withtritiated lecuine (³H-leucine) and SP-PE toxin (made as described inExample 1). Viable cells incorporate the amino acid leucine topolypeptides. Dead or dying cells do not synthesize polypeptide and donot incorporate the ³H-leucine. FIG. 1 shows a schematic summary of dataof experiments testing the ability of the SP-PE conjugate toxin of theinvention to kill CHO cells stably transfected with and expressing NK-1receptors (FIG. 1A), NK-2 receptors (FIG. 1B), and NK-3 receptors (FIG.1C).

The SP-PE toxin only comprised the viability of NK-1 receptor-expressingcells. The viability of NK-1 and NK-3 receptor expressing cells was thesame as the (negative) control. As a (negative) control, in place ofSP-PE toxing, the cells were exposed to the PE domain alone (lacking thereceptor ligand SP) (designated “PE35” in FIG. 1). PE domain alone didnot comprise the viability of any of the transfected cells.

These data demonstrate that the SP-PE toxin conjugates of the inventionspecifically ablate NK-1 receptor expressing cells.

Example 2 SP-Pseudomonas Exotoxin Disulfide Linked Conjugate KillsNK-1-Expressing Cells in Rat Spinal Column Dorsal Horn Cells In Vivo

The following example details a study which demonstrates thatcompositions and methods of the invention can be practiced to ablateNK-1 receptor expressing cells in the dorsal horn of the spinal columnin vivo. Specifically, this art-recognized animal model for pain therapydemonstrates that the SP-PE toxin of the invention administered in vivovia intrathecal injection selectively and potently killed dorsal hornNK-1 receptor expressing cells. The toxin conjugate of the invention wasextremely effective deleting NK-1 receptor expressing cells from thedorsal horn of the spinal cord and NK-1 receptor expressing cells fromthe striatum.

Male Sprague-Dawley rats were used in all experiments. All proceduresand experimental protocols were approved by an NIH Animal Care and UseCommittee and are in accordance with the guidelines of the InternationalAssociation for the Study of Pain (Zimmermann (1983) Pain 16:109-110).Animals were anesthetized and the SP-PE toxin of the invention (asdescribed in Example 1, above) administered by intrathecal infusion asdescribed by Benoliel (1999) Pain 79:243-253, at a dose of 20 μlcontaining a concentration of 25 nanograms per μl

Tissue preparation and immunocytochemistry were performed as by Benoliel(1999) supra. Tissue sections were harvested eight days afterintrathecal treatment with SP-PE toxin (or saline as a negativecontrol). Dorsal horn tissue sections of the spinal cord were stainedwith antibodies for NK-1 receptor. Sections from animals treated withSP-PE toxin clearly showed significant ablation of SPreceptor-expressing cells (loss of anti-NK-1 receptor antibodyreactivity) in the dorsal horn of the rat spinal column. Animals treatedwith saline only showed normal SP receptor-expressing cell stainingpattern, i.e., no ablation. The saline control showed strong stainingfor an antibody reactive NK-1 receptor.

These data demonstrate that the SP-PE toxins of the invention ablateNK-1 receptor expressing cells in the dorsal horn of the spinal columnin vivo.

Example 3 SP-Pseudomonas Exotoxin Disulfide Linked ConjugateAdministered In Vivo Decreased Pain Sensation in a Heat HyperalgesiaTest

The following example details a study with an art-recognized animal(rat) model for investigating treatments for pain which shows that theSP-PE compositions of the invention can be used and methods of theinvention can be practiced to treat pain.

All animal procedures and intrathecal toxin administrations were asdescribed above (Example 2), as in Benoliel (1999) supra. The SP-PEtoxin used was prepared as described in Example 1, above. Heatsensitivity was assayed with a withdrawal latency test as described byIadarola (1988) Brain Res. 455:205-212, see also Benoliel (1999) supra.Sensitivity to nociceptive thermal stimulation was tested inunrestrained rats with a radiant heat stimulus as described. Briefly,the rat is placed on an elevated glass plate, with a clear plastic cageinverted over the animal. After 5 min habituation to the enclosure, aprojector lamp in a conical housing is positioned under the fore or hindpaws or tail. The light emerges from an opening in the conical housinglight. The latency is recorded automatically when paw or tail movementinterrupts the readings of a photocell in the lamp housing. Four ratswere tested with toxin and four rats were controls. Assays wereconducted 20 days after toxin treatment.

FIG. 2 shows a schematic summary of data of experiments testing theability of the SP-PE conjugate toxin of the invention to treat pain.Administration of SP-PE toxin significantly increased paw withdrawallatency as compared to control (saline only administered). The “normal”or saline control latency was 8 seconds while the “pain desensitized”toxin treated animal had a withdrawal latency of between 14 and 16seconds. Similar effects are also observed at 8 days following SP-PEadministration.

To further evaluate long-term effects and selectivity following SP-PEtreatment, control and treated animals were analyzed for withdrawallatency. heat withdrawal latency was tested as decribed in Example 2.

The ability of SP-PE to selectively block thermal heat pain in rats at atarget region was tested 46 days after intrathecal injection of about 15picomoles of SP-PE (FIG. 3), which was infused slowly at a rate of about1 μl per minute (dose of 20 μl containing a concentration of 25nanograms per μl). In this study, the conjugate was administerd to theregion of the cord that affects pain perception in the tail. The resultsshow segmental selectivity of SP-PE applied at sacral spinal cord anddemonstrate that the effect of SP-PE treatment can be localized tocontrol the site of action, even though the injection is made into afluid-filled space.

To demonstrate that administration of SP-PE to rats attenuates a symptomof chronic pain, hyperalgesia, a rat thermal hyperalgesia model wasused. This method measures cutaneous hyperalgesia to thermal stimulationin unrestrained animals, and was performed essentially as described byHargreaves (1988) Pain 32:77-88. Briefly, after administration ofexpression vector, baseline thermal hyperalgesia is assessed by pawwithdrawal latency to thermal stimulus. Inflammation is then inducedwith unilateral injection of 6 mg carrageenan (type IV Sigma C-3889) involum of 0.15 mL PBS into a hindpaw of a 200-300 g male rat. Pawwithdrawal latency to thermal stimulus is again measured on both theinflamed and uninflamed paws.

The ability of SP-PE to block acute thermal pain in the foot and toblock thermal hyperalgesia in the carrageenan model of hind pawinflammation was tested at 46 days following treatment (FIG. 4). Thedata obtained from the animals before induction of inflammatin, (thepre-inflamed data) show that 46 days after intrathecal injection of 15picomoles SP-PE, the treated animals showed an increased withdrawallatency. When tested in the inflammation model system, the treatedanimals also showed blocking of thermal hyperlgesia, which incates thatthe SP-PE will also be useful in chronic pain syndromes.

Treatment with SP-PE also blocks mechanical pain, i.e., a mild pinchwith a toothed forceps. This effect occurs in a segmental fashion. Thus,if the infusion occurs around the hindpaw region of the spinal cord, theloss of pinch sensitivity is seen in the tail, hind paws, and part ofthe lower trunk, but not in the upper half of the body.

This art-recognized animal (rat) model for investigating treatments forpain clearly demonstrate that the SP-PE toxin conjugates of theinvention are effective in the treatment of pain in vivo.

Example 4 Long Term Effects On Other Neurotransmitter Systems FollowingSP-PE Treatment

This example shows that other neurotransmitter systems do not undergolong term compensatory rearrangements following treatment with SP-PE.

Compensatory rearrangements of the nervous system might occur afterremoval of a cell population, which, in turn could alter the therapeuticeffects of administration of SP-PE. To evaluate the effects of SP-PEtreatment on other neurotransmitter systems, immunocytochemistry forcalcitonin gene-related peptide (CGRP) and tyrosine hydroxylase (TH) wasperformed on dorsal horn tissue samples obtained from control and SP-PEtreated animals at 20 days post treatment. Tissue samples were preparedas in Example 2 and sections were stained with antibodies specific forCGRP or TH.

The distribution of CGRP, which is produced by primary afferent neurons,was analyzed 20 days following administeration of 15 picomoles SP-PE.The results showed that the distribution and amount of CGRP observed intissue samples in the treated animals appeared to be equivalent to thatof the control.

Tyosine hydroxylase is involved in the production of the biogenicamines, dopamine, norepinephrine (the transmitter in sympathetic nervoussystem fibers), and epinephrine. These neurotransmitters have beenimplicated in rearrangements in the dorsal root ganglion upon nerveinjury. The result of the immunocytochemistry analysis at 20 days posttreatment showed no consistent effects on TH innervation in treatedanimals, which is likely due to the fact that only a small population ofcells is being removed from the dorsal spinal cord.

These experiments demonstrate that administration of SP-PE does notresult in compensatory rearrangement. The magnitude of the decrease insensitivity to pain in SP-PE treated animals was also equivalent at 70days after administration of the conjugate to that observed at 20-46days post-treatment. Further, animals at over 70 days post-treatmentexhibited normal locomotion and there was no evidence of autotomy.

Example 5 Intrastriatal Administration of SP-PE

The following example shows that intrastriatal adminstration of SP-PEresults in deletion of NK-1 expressing striatal neurons.

Rats administered SP-PE instrastriatally received a atotal dose of 25 ngof the conjugate. Animals were anesthetized and a one μl injection ofconjugate was made over about 1 minute into the striatum. The rats werethen assessed for apomorphine-induced turning behavior and neuronaltoxicity.

A unilateral intrastriatal injection of SP-PE did not produce anappreciable alteration in the rats locomotor activity, posture or gait.However, when challenged with a systemically administered apomorphine,the rats displayed a strong contralateral turning behavior. Apomorphinealso produce hyper-locomotion.

Immunocytochemical analysis also showed that the same dose that producedthe contralateral turning behavior also produce a marked decrease in NK1immunoreactive neurons in the rat striatum. The baseline NK1immunoreactivity was most dense in the dorsal and lateral straitum andwas associated with the dendrites of the neurons, although in theventral striatum and olfactory tubercle, neuronal cell bodies wereclearly observed. Previous research has shown that the NK1 positivestriatal neurons are the medium spiny cholinergic interneurons.Microinjection of 25 ng of SP-PE (˜0.7 picomoles) produced amacroscopically noticeable, unilateral zone of NK1 receptor loss instriatum, in comparison to the contralateral side of the same animal.The neuropil on the non-injected side contained a dense amount ofNK1-immunopositive dendrites and cell bodies. These were eliminated fromthe injected zone on the other side.

The direct intrastriatal injections also provided an opportunity toassess potential toxic effects on non-receptor expressing cells exposeddirectly to the SP-PE conjugate. The integrity of other neural elementswas assessed in two ways. First, the NK1 stained sections wereimmunocytochemically counter-stained for neurofilament protein, whichlocalizes to the cortico-spinal fiber bundles. The bundles were are notaffected by SP-PE even though they are in the center of the zone ofmedium spiny interneuron denervation. Second, standard histologicalstains of paraffin embedded thin sections were used to assesspathological changes. The Sections that included the cannula tract werestained with hematoxylin-eosin or luxol-fast blue. If a large amount ofnon-specific cell death had occurred a widespread loss of neuronalperikarya and a large microglial inflitration surrounding the tip of thecannula could be expected. Neither effect was observed, the appearanceof the injected srtiatum was indistinguishable from either surroundingtissue or the contralateral side in which the NK1 receptor bearing cellswere unaffected.

Thus, instrastriatal administration of SP-PE produced behavioral effects(contraversive turning with a apomorphine challenge, 3 mg/kgintraperitoneal injection) and selective loss of SPR expressingcholinergic interneurons with no concurrent loss of Mu-opioid receptors.

Example 6 SP-Pseudomonas Exotoxin Disulfide Linked ConjugateAdministered To Patients with Chronic Pain

The following example details the treatment of chronic pain in patientsusing the SP-PE compositions, e.g., the pharmaceuticals, of theinventions.

As substance P (SP_ peptide is a known mediator of chronic pain (as aligand for the NK-1 recptor), the SP-PE pharmaceuticals of the inventioncan be efficaciously administered to virtually any patient havingchronic pain. Means to diagnose and assess chronic pain is discussed indetail, above. As noted above, while chronic pain is diagnosed andtreatments are assessed using both objection and subjective criteria, adiagnosis of a particular pathologic process (which is usually alsochronic) known to produce or be associated with chronic pain is helpfulto the clinician in determining when the administer the pharmaceuticalcompositions of the invention. Such conditions are well characterizedand include, e.g., chronic pain syndrome, arthralgia, osteoarthritis andrheumatoid arthritis, causalgia, hyperpathia, neuralgia, neuritis,radiculagia, fibromyalgia, orofacial pain and temporomandibulardisorders, reflex sympathetic dystrophy, chronic back pain, certaincancers, and the like.

In one exemplary protocol, patients diagnosed with chronic pain aretreated with an SP-PE pharmaceutical compositions suitable forintrathecal administation. While the dosage schedule, i.e., the “dosingregimen,” will depend upon a variety of factors, including, e.g., theamount of chronic pain present, the duration of the pain, the stage andseverity of the disease or condition associated with the chronic pain(if any), and the general state of the patient's health, physicalstatus, age and the like, one typical dosage is between about 0.5 toabout 50 mL of a formulation at dosages equivalent to about 0.2 to about0.6 mg/kg daily for five days. This is administered by an internal orexternal intrathecal infusion pump. These pumps, while typically usedfor administration of opioids, can be used to administer thepharmaceuticals of the present invention, see, e.g., Likar (1999)Arzneimittelforschung 49:489-493; Valentino (1998) J. Neurosci. Nurs.30:233-239, 243-234.

Dosage levels and frequency of administration are continuously adjustedbased on objective and subjective criteria, as discussed herein. Anydosage can be used as required and tolerated by the patient.Furthermore, the exact concentration of toxin conjugate, the amount offormulation, and the frequency of administration can be adjusteddepending on the levels of SP-PE measured in the CSF after an initialadministration. Means to sample CSF or other fluid or tissue samples anddetect and quantitate levels of SP-PE are discussed above.

1. A method of making a cell toxin comprisng reacting a polypeptidecomprising a Pseudomonas exotoxin translocation domain linked to aPseudomonas exotoxin ADP-ribosylatin domain, wherein the Pseudomonasexotoxin translocation domain comprises at least one reactive sulfhydrylgroup, with a substance P peptide comprising one additional cysteineresidue at its amino terminal end, wherein the cysteine sulfhydryl groupis disulfide linked to a di-thiobis (2-nitro)-benzoic acid group, suchthat after the reaction a disulfide bond is formed between the exotoxinpolypeptide and the substance P and a thionitrobenzoate group isreleased, and purifying the substance P-Pseudomonas exotoxin disulfidelinked conjugate from the released thionitrobenzoate group such that thepurified conjugate is substantially free of thionitrobenzoate groups. 2.The method of claim 1, wherein the Pseudomonas exotoxin translocationdomain sulfhydryl group located within ten amino acid residues of thetranslocation domain amino terminus.
 3. The method of claim 1, whereinthe Pseudomonas exotoxin translocation domain sulfhydryl group locatedat the translocation domain amino terminus.
 4. The method of claim 1,wherein the Pseudomonas exotoxin translocation domain sulfhydryl groupis a cysteine residue.
 5. The method of claim 1, wherein the Pseudomonasexotoxin translocation domain is covalently linked to the Pseudomonasexotoxin ADP-ribosylation domain.
 6. The method of claim 5, wherein thecovalent linkage between the Pseudomonas exotoxin translocation domainand the Pseudomonas exotin ADP-ribosylation domain is a peptide bond. 7.The method of claim 1, wherein the Pseudomonas exotoxin translocationdomain comprises an amino acid sequence as set forth in SEQ ID NO:1 andthe Pseudomonas exotoxin ADP-ribosylation domain comprises an amino acidsequence as set forth in SEQ ID NO:2.
 8. A pharmaceutical compositionfor the ablation of NK1 receptor expressing cells comprising a celltoxin and a pharmaceutically acceptable excipient, wherein the celltoxin is a substance P-Pseudomonas exotoxin disulfide linked conjugatemade by a process comprising the following steps: reacting a polypeptidecomprising a Pseudomonas exotoxin translocation domain linked to aPseudomonas exotoxin ADP-ribosylation domain, wherein the Pseudomonasexotoxin translocation domain comprises at least one reactive sulfhydrylgroup, with a substance P peptide comprising one additional cysteineresidue at its amino terminal end, wherein the cysteine sulfhydryl groupis disulfide linked to a di-thiobis (2-nitro)-benzoic acid group, suchthat after the reaction a disulfide bond is formed between the exotoxinpolypeptide and the substance P and a thionitrobenzoate group isreleased, and purifying the substance P-Pseudomonas exotoxin disulfidelinked conjugate from the released thionitrobenzoate group such that thepurified conjugate is substantially free of thionitrobenzoate groups. 9.The pharmaceutical composition of claim 8, wherein the Pseudomonasexotoxin translocation domain sulfhydryl group located within ten aminoacid residues of the translocation domain amino terminus.
 10. Thepharmaceutical composition of claim 8, wherein the Pseudomonas exotoxintranslocation domain sulfhydryl group located at the translocationdomain amino terminus.
 11. The pharmaceutical composition of claim 8,wherein the Pseudomonas exotoxin translocation domain sulfhydryl groupis a cysteine residue.
 12. The pharmaceutical composition of claim 8,wherein the Pseudomonas exotoxin translocation domain is covalentlylinked to the Pseudomonas exotoxin ADP-ribosylation domain.
 13. Thepharamceutical composition of claim 12, wherein the covalent linkagebetween the Pseudomonas exotoxin translocation domain and thePseudomonas exotoxin ADP-ribosylation domain is a peptide bond.
 14. Thepharmaceutical composition of claim 8, wherein the Pseudomonas exotoxintranslocation domain comprises an amino acid sequence as set forth inSEQ ID NO:1 and the Pseudomonas exotoxin ADP-ribosylation domaincomprises an amino acid sequence as set forth in SEQ ID NO:2.
 15. Thepharmaceutical composition of claim 8, wherein the cell toxin andpharamceutically acceptable excipient are suitable for administrationintrathecally, subdurally or directly into the brain parenchyma.
 16. Amethod for ablating an NK1 receptor expressing cell in a patientcomprising administering to said patient a cell toxin in apharmaceutically acceptable excipient in an amount sufficient to ablatean NK1 receptor expressing cell, wherein the cell toxin is a substanceP-Pseudomonas exotoxin disulfide linked conjugate made by a processcomprising the following steps: reacting a polypeptide comprising aPseudomonas exotoxin translocation domain linked to a Pseudomonasexotoxin ADP-ribosylation domain, wherein the Pseudomonas exotoxintranslocation domain comprises at least one reactive sulfhydryl group,with a substance P peptide comprising one additional cysteine residue atits amino terminal end, wherein the cysteine sulfhydryl group isdisulfide linked to a di-thiobis (2-nitro)-benzoic acid group, such thatafter the reaction a disulfide bond is formed between the exotoxinpolypeptide and the substance P and a thionitrobenzoate group isreleased, and purifying the substance P-Pseudomonas exotoxin disulfidelinked conjugate from the released thionitrobenzoate group such that thepurified conjugate is substantially free of thionitrobenzoate groups.17. The method of claim 16, wherein the ablated NK1 receptor expressingcell is a dorsal horn cell, a stratum cell or a brain parenchyma cell.18. A method of treating chronic pain without significantly afftectingbasal nociceptive responses comprising administering to a subject inneed thereof a cell toxin in a pharmaceutically acceptable excipient inan amount sufficient to treat chronic pain without significantlyafftecting basal nociceptive responses, wherein the cell toxin is asubstance P-Pseudomonas exotoxin disulfide linked conjugate made by aprocess comprising the following steps: reacting a polypeptidecomprising a Pseudomonas exotoxin translocation domain linked to aPseudomonas exotoxin ADP-ribosylation domain, wherein the Pseudomonasexotoxin translocation domain comprises at least one reactive sulfhydrylgroup, with a substance P peptide comprising one additional cysteineresidue at its amino terminal end, wherein the cysteine sulfhydryl groupis disulfide linked to a di-thiobis (2-nitro)-benzoic acid group, suchthat after the reaction a disulfide bond is formed between the exotoxinpolypeptide and the substance P and a thionitrobenzoate group isreleased, and purifying the substance P-Pseudomonas exotoxin disulfidelinked conjugate from the released thionitrobenzoate group such that thepurified conjugate is substantially free of thionitrobenzoate groups.19. The method of claim 18, wherein cell toxin is administered toepineurium cells, perineurium cells, nerve ganglia, nerve sheathers,nerve linings, meninges, pia mater cells, arachnoid membrane cells, duramembrane cells, cells lining a joint or brain or spinal cord parenchymalcells.